The present invention relates to methods and compositions for increasing the production of high titre stocks of recombinant AAV (rAAV) through regulation of expression of the AAV REP proteins. The methods and compositions of the invention are based on the observation that low level expression of the AAV REP protein increases the efficiency of rAAV DNA replication and the production of AAV viral capsid protein resulting in production of higher titre recombinant viral stocks. The invention encompasses methods and compositions for controlling the level of REP expression at the transcriptional or translational level. Additionally, the invention provides methods for regulating the biological activity and/or stability of the REP proteins at the post-translational level. The methods and compositions of the invention can be used to produce high titre stocks of rAAV which can be used in gene therapy for the purpose of transferring genetic information into appropriate host cells for the management and correction of human diseases including inherited and aquired disorders.
Gene therapy is generally understood to refer to techniques designed to deliver functionally active therapeutic genes into targeted cells. Such therapeutic genes may encode proteins that complement genetic deficiencies, cytokines, cell surface membrane proteins or any protein that functions to regulate cell growth and/or differentiation. Such proteins may function intracellularly, for example, by regulating a signalling pathway or transcriptional pathway. Alternatively, the proteins may be secreted by the cell and exert their effect extracellularly.
Initial efforts toward somatic gene therapy have relied on indirect means of introducing genes into tissues, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and reimplanted into the body. These types of techniques are generally referred to as in vitro treatment protocols.
In addition, recombinant replication-defective viral vectors have been used to infect cells both in vitro and in vivo. Perhaps the most widely studied viral vectors for use in gene therapy have been the retroviral vectors. The major disadvantages-associated with the use of retroviral vectors include the inability of many viral vectors to infect non-dividing cells, problems associated with insertional mutagenesis and potential helper virus production. Recently, attention has turned to the use of other types of recombinant viral vectors such as adenovirus and adeno-associated virus based vectors, that may be used to deliver genes of interest to cells.
In particular, recombinant adeno-associated virus has many features of interest in the field of gene therapy. The vectors are based on a defective, nonpathogenic human parvovirus that can infect both dividing and non-dividing cells without a marked tropism. In addition, the viral genome can stably integrate within the host genome, facilitating long term gene transfer.
The AAV genome is composed of a linear single stranded DNA molecule of 4680 nucleotides which contains major open reading frames coding for the Rep (replication) and Cap (capsid) proteins. Flanking the AAV coding regions are two 145 nucleotide inverted terminal (ITR) repeat sequences that contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. In addition, the ITR sequences are needed for viral integration, rescue from the host genome and encapsidation of viral nucleic acid into mature virions (Muzyczka, N., 1992, Current Topics in Microbiology and Immunology. 158, 97-129).
AAV can assume two pathways upon infection into the host cell depending on whether helper virus is present. In the presence of helper virus, AAV will enter the lytic cycle whereby the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome will integrate as a provirus into a specific region of the host cell genome through recombination between the AAV termini and host cell sequences (Cheung, A. et al., 1980, J. Virol. 33:739-748; Berns, K. I. et al., 1982, in Virus Persistence, eds. Mahey, B. W. J., et al. (Cambridge Univ. Press, Cambridge), pp. 249-265).
The use of AAV as a vehicle for the transfer of genetic information has been facilitated by the discovery that when a plasmid containing an intact AAV genome is transfected into a host cell the recombinant AAV vector will integrate into the host cell genome and remain as a provirus until the host cell subsequently becomes infected with a helper virus. Upon infection of the host cell with helper virus, the AAV is rescued out from the plasmid vector and enters the lytic cycle leading to production of mature virions.
The production of rAAV particles, utilizes a vector containing a transgene flanked by the inverted terminal repeats (ITR), which are the sole AAV cis sequences required for DNA replication, packaging and integration. To produce rAAV particles, the AAV (Rep) and capsid (Cap) gene products are normally provided in trans from a different template, usually a helper plasmid.
The three viral coat proteins, VP1, VP2, and VP3 which are required for virion expression are derived from mRNA initiated at the p40 promoter, while the four overlapping non-structural Rep proteins are essential for AAV DNA replication. Rep78 and 68 are expressed from unspliced and spliced transcripts initiating at the p5 promoter, while Rep52 and Rep40 are similarly produced from transcripts initiating at the p19 promoter. Although Rep52/40 have been implicated in AAV single stranded DNA formation (Chejanovsky et al., 1989, Virology 173:120-128) and gene regulation, Rep appear to display all enzyme functions essential for AAV DNA replication, (ITR binding, DNA helicase, and DNA site-specific nicking activity), (Muzyczka, N., 1991, Seminars in Virology 2:281-290). In addition to these functions, Rep both positively and negatively regulate AAV promoters (Labow et al., 1986, Journal of Virology 60:215-258; Pereira et al., 1997, J. Virol, In Press; Tratschin et al., 1986, Mol. Cell Biol. 6:2884-2894) and repress numerous heterologous promoters (Antoni et al., 1991, Journal of Virology 65:396-404; Heilbronn et al., 1990, Journal of Virology 64:3012-3018; Hermonat, P. L., 1994, Cancer Letters 81:129-36; Horer, et al., 1995, Journal of Virology 69:5485-5496; Labow et al., 1987, Molecular and Cellular Biology 7:1320-1325).
Rep gene expression appears to be critical for all steps of the AAV life cycle, including a latent state which occurs in the absence of a helper virus (Berns, K. I., 1990, Virology, 2ed, vol. 2; Berns, K. I., 1996, B. N. Fields et al. ed.; Samulski et al., 1989, Journal of Virology 63:3822-3828). Recently, Rep have also been associated with AAV site-specific integration (Giraud et al., 1994, Proceedings of the National Academy of Sciences of the United States of America; Kotin et al., 1990, Proceedings of the National Academy of Sciences of the United States of America 87:221-2215; Samulski et al., 1991, EMBO Journal 10:3941-3950; Weitzmann et al., 1994, Proceedings of the National Academy of Sciences of the United States of America 91:5808-5812). Repression of viral gene expression by rep and host YY1 protein appears to be required for establishment and maintenance of the latent state (Labow et al., 1986, Journal of Virology 60:251-258; Laughlin et al., 1982, Journal of Virology 41:868-876; Periera et al., 1997, J. Virol In Press; Shi et al., 1991, Cell 67:377-388). Such repression may be necessary to avoid the demonstrated cytostatic effect on the host cell by Rep gene products (Yang et al., 1994, Journal of Virology 68:4847-4856). During a lytic infection, the AAV promoters, particularly p5, are transactivated by the adenovirus ElA proteins and YY1 (Lewis, et al., 1995, J. Virol. 69:1628-1636; Shi et al., 1991, Cell. 67:377-388). The p5 products then positively regulate the p19 and p40 promoters, resulting in abundant production of Rep 52/40 and viral capsid proteins (Pereira et al., 1997, J. Virol. In Press). Early effort to by-pass AAV rep gene regulation by substituting the p5 promoter with the SV40 early promoter failed (Labow et al., 1988, Journal of Virology 62:1705-1712). Instead of constitutive Rep expression, the heterologous promoter unexpectedly behaved in the same manner as the endogenous p5 promoter; repressed in the absence and activated in the presence of Ad (Labow et al., 1988, Journal of Virology 62:1750-1712). While these studies were the first to suggest rep repression as a mechanism for regulating heterologous promoters, these findings also implied that AAV p5 products may be a rate-limiting factor in AAV production (Labow et al., 1988, Journal of Virology 62:1705-1712). Further efforts in this area have suggested that overexpression of Rep may increase rAAV vector yields (Flotte et al., 1995, Gene Therapy 2:29:37).
An essential feature for use of rAAV as an efficient delivery system is the ability to produce recombinant stocks of virus. Although rAAV titres can approach wild type (wt) levels after multiple rounds of purification and concentration, the overall total yield is still substantially lower than that of wild type AAV. Therefore, methods that increase the ability to produce high titre rAAV viral stocks will facilitate the use of rAAV delivery systems in gene therapy.
The present invention provides methods and compositions for increasing the production of high titre stocks of recombinant AAV. The invention is based on the discovery that decreased expression of AAV REP proteins results in increased synthesis of viral capsid proteins and replication of viral DNA resulting in production of high titre recombinant viral stocks. Such recombinant AAV stocks may be used in gene therapy for the purpose of transferring genetic information into appropriate host cells for the management and correction of human disease including inherited and acquired disorders such as cancer and AIDS.
The invention encompasses methods for increasing the production of high titre stocks of recombinant AAV by regulating the expression levels and/or activity of the AAV REP proteins in a host cell. The invention further encompasses compositions such as recombinant helper plasmids that are genetically engineered to express low levels of biologically functional viral REP proteins. In such helper plasmids the expression of REP proteins may be regulated at the transcriptional, translational and/or post-translational level.
The expression of REP proteins may be regulated at the transcriptional level through the use of tightly controlled promoter systems that result in either low level, or inducible, expression of the REP gene. Such promoters can be genetically engineered into recombinant helper plasmids that are designed to express low levels of REP protein. Further, triple helix molecules can be utilized to reduce the level of REP gene expression. Such triple helix molecules can be designed to hybridize to the promoter region of the REP gene and thereby inhibit REP gene transcription.
Further, the invention encompasses the coding region of the REP genes which are genetically engineered to replace the initiator MET codon with a less efficiently translated initiator codon. The genes encoding the viral REP proteins of the present invention can also be genetically engineered to contain specific 5xe2x80x2 nucleotide sequences to which translation repressor proteins bind. The binding of such repressor proteins to the 5xe2x80x2 end of the REP mRNA molecules will result in inhibition of REP mRNA translation. Using such a system the level of REP protein can be controlled by regulating the level and/or activity of the translational repressor protein in the host cell.
Alternatively, the level of REP expression may also be controlled altering the stability of REP mRNA. For example, the half life of the REP mRNAs may be significantly decreased by genetically engineering nucleotide sequences rich in A and U nucleotides in the 3xe2x80x2 untranslated region (UTR). Additionally, REP mRNAs containing recognition sites in their 3xe2x80x2 UTR for specific endonucleases may be generated using recombinant DNA techniques.
The level of REP protein may be controlled by taking advantage of a translational process referred to as translational recoding. In such a process, a specific recording signal in the mRNA molecule causes the growing polypeptide chain occassionally to slip backward by one nucleotide on the ribosome as translation proceeds, causing the mRNA to be read in the incorrect reading frame.
The level of REP protein expressed in a host cell may further be reduced through the use of antisense and ribozyme molecules. Antisense approaches involve the design of oligonucleotides that bind to the complementary REP RNA and suppress translation of REP RNA. Ribozymes molecules may be designed that include one or more sequences complementary to REP RNA and which function to specifically and efficiently catalyses endonucleolytic cleavage of REP RNA sequences.
Finally, mutant forms of the REP proteins may be generated that have decreased activity and/or decreased protein stability. The activity of the REP proteins may be regulated through the use of temperature sensitive REP mutants. Alternatively, REP proteins which are less stable, i.e., REP proteins that possess a shorter half-life or REP proteins that are more susceptible to proteolytic cleavage, may be utilized as a means for decreasing the activity of the REP proteins.
The invention is demonstrated by way of examples, in which the overexpression of the REP gene was shown to inhibit rAAV DNA replication and CAP gene expression. In contrast, reduced production of AAV REP protein expression was sufficient for production of higher titres of recombinant virus.